Biological membranes surround all cells and mediate all their interactions with the outside world. Depending on the biological context, membrane proteins act as receptors, enzymes, channels, transporters, structural proteins and cell adhesion molecules and, as such, contribute to a wide variety of essential cellular functions. We propose to establish the Transcontinental EM Initiative for Membrane Protein Structure as a PSI: Biology Center for Membrane Protein Structure Determination. Based on the biological interests of our participants and their collaborators, we have selected a group of mostly eukaryotic targets that play important roles in human biology and disease. In particular, our targets are involved in membrane transport (aquaporin, TRP and GIRK channels, ion pumps, transporters for drugs and heme), in signaling (G-protein couple receptors, intramembrane proteases and bacterial two-component systems), and in cell adhesion (aquaporin and the MP20 tetraspanin from the lens). Many of these targets form complexes, either between membrane-bound subunits or with soluble partners, and therefore represent a significant challenge to conventional methods of structure determination (i.e., X-ray and NMR). We will use cryo-electron microscopy (cryo-EM) as our tool for structure determination, primarily by growing two-dimensional crystals within a lipid bi-layer but also by imaging isolated complexes within detergent micelles. Indeed, cryo-EM has an established track record in structure determination at atomic resolution and offers advantages for membrane proteins by providing fewer crystallization constraints and a native membrane environment. For our Center, we have brought together four investigators with extensive experience in electron crystallography to establish high-throughput methods for screening and optimizing 2D crystallization. A fifth investigator has a strong track record in computation cryo-EM and will spearhead efforts to develop novel methods for structure determination. We are convinced that by applying high-throughput methods to 2D crystallization and by modernizing our methods for structure determination, cryo-EM can make a substantial contribution to our understanding of membrane protein biology. PUBLIC HEALTH RELEVANCE: Membranes surround all cells and proteins within this membrane mediate the flow of information and materials. As a result, membrane proteins are implicated in many diseases. Structural information about these proteins is critical to understanding the biology behind the disease and for designing drugs to ameliorate the problems.